CRISPR/Cas9-Targeted Mutagenesis of CiGAS and CiGAO to Reduce Bitterness in Chicory (Cichorium intybus L.).

BACKGROUND: Chicory (Cichorium intybus L.), a member of the Asteraceae family, is known for its numerous health benefits, including its prebiotic, digestive, antioxidant or anti-inflammatory effects. Used as a coffee substitute, chicory roots is also appreciated for its bitterness, which can prove to be a disadvantage for other uses in food. The bitterness of chicory is largely linked to the presence of sesquiterpene lactones (STLs) in the roots. METHODS: In order to create less bitter industrial chicory varieties, CRISPR/Cas9 technology was used to inhibit the first two genes of the STL biosynthetic pathway: germacrene A synthase (CiGAS), short form, and germacrene A oxidase (CiGAO). To determine the impact of these reductions on the perception of bitterness, a sensory analysis of 13 field-grown chicories genotypes, contrasting for their STL composition, allowed the construction of obtain a bitterness scale by correlating STL content with perceived bitterness. The edited chicories were positioned on this scale according to their STL content. RESULTS: Biallelic mutations in two of the copies of CiGAS-short form or in the CiGAO gene led to a reduction in STL content of edited chicories and a reduction in bitterness, or even an absence of perception, was obtained for some mutants. CONCLUSIONS: The use of the CRISPR/Cas9 tool as well as the choice of targets therefore makes it possible to modulate the bitterness of chicory.

Genome-Wide Datasets of Chicories (Cichorium intybus L.) for Marker-Assisted Crop Breeding Applications: A Systematic Review and Meta-Analysis.

Cichorium intybus L. is the most economically important species of its genus and among the most important of the Asteraceae family. In chicory, many linkage maps have been produced, several sets of mapped and unmapped markers have been developed, and dozens of genes linked to traits of agronomic interest have been investigated. This treasure trove of information, properly cataloged and organized, is of pivotal importance for the development of superior commercial products with valuable agronomic potential in terms of yield and quality, including reduced bitter taste and increased inulin production, as well as resistance or tolerance to pathogens and resilience to environmental stresses. For this reason, a systematic review was conducted based on the scientific literature published in chicory during 1980-2023. Based on the results obtained from the meta-analysis, we created two consensus maps capable of supporting marker-assisted breeding (MAB) and marker-assisted selection (MAS) programs. By taking advantage of the recently released genome of C. intybus, we built a 639 molecular marker-based consensus map collecting all the available mapped and unmapped SNP and SSR loci available for this species. In the following section, after summarizing and discussing all the genes investigated in chicory and related to traits of interest such as reproductive barriers, sesquiterpene lactone biosynthesis, inulin metabolism and stress response, we produced a second map encompassing 64 loci that could be useful for MAS purposes. With the advent of omics technologies, molecular data chaos (namely, the situation where the amount of molecular data is so complex and unmanageable that their use becomes challenging) is becoming far from a negligible issue. In this review, we have therefore tried to contribute by standardizing and organizing the molecular data produced thus far in chicory to facilitate the work of breeders.

Insights into the convergent evolution of fructan biosynthesis in angiosperms from the highly characteristic chicory genome.

Fructans in angiosperms play essential roles in physiological functions and environmental adaptations. As a major source of industrial fructans (especially inulin-type), chicory (Cichorium intybus L.) is a model species for studying fructan biosynthesis. However, the genes underlying this process and their evolutionary history in angiosperms remain elusive. We combined multiple sequencing technologies to assemble and annotate the chicory genome and scan its (epi)genomic features, such as genomic components, DNA methylation, and three-dimensional (3D) structure. We also performed a comparative genomics analysis to uncover the associations between key traits and gene families. We achieved a nearly complete chicory genome assembly and found that continuous bursts of a few highly active retrotransposon families largely shaped the (epi)genomic characteristics. The highly methylated genome with its unique 3D structure potentially influences critical biological processes. Our comprehensive comparative genomics analysis deciphered the genetic basis for the rich sesquiterpene content in chicory and indicated that the fructan-accumulating trait resulted from convergent evolution in angiosperms due to shifts in critical sites of fructan-active enzymes. The highly characterized chicory genome provides insight into Asteraceae evolution and fructan biosynthesis in angiosperms.

The genomes of chicory, endive, great burdock and yacon provide insights into Asteraceae palaeo-polyploidization history and plant inulin production.

Inulin is an important reserve polysaccharide in Asteraceae plants, and is also widely used as a sweetener, a source of dietary fibre and prebiotic. Nevertheless, a lack of genomic resources for inulin-producing plants has hindered extensive studies on inulin metabolism and regulation. Here, we present chromosome-level reference genomes for four inulin-producing plants: chicory (Cichorium intybus), endive (Cichorium endivia), great burdock (Arctium lappa) and yacon (Smallanthus sonchifolius), with assembled genome sizes of 1.28, 0.89, 1.73 and 2.72 Gb, respectively. We found that the chicory, endive and great burdock genomes were shaped by whole genome triplication (WGT-1), and the yacon genome was shaped by WGT-1 and two subsequent whole genome duplications (WGD-2 and WGD-3). A yacon unique whole genome duplication (WGD-3) occurred 5.6-5.8 million years ago. Our results also showed the genome size difference between chicory and endive is largely due to LTR retrotransposons, and rejected a previous hypothesis that chicory is an ancestor of endive. Furthermore, we identified fructan-active-enzyme and transcription-factor genes, and found there is one copy in chicory, endive and great burdock but two copies in yacon for most of these genes, except for the 1-FEH II gene which is significantly expanded in chicory. Interestingly, inulin synthesis genes 1-SST and 1-FFT are located close to each other, as are the degradation genes 1-FEH I and 1-FEH II. Finally, we predicted protein structures for 1-FFT genes to explore the mechanism determining inulin chain length.

Consecutive action of two BAHD acyltransferases promotes tetracoumaroyl spermine accumulation in chicory.

Fully substituted phenolamide accumulation in the pollen coat of Eudicotyledons is a conserved evolutionary chemical trait. Interestingly, spermidine derivatives are replaced by spermine derivatives as the main phenolamide accumulated in the Asteraceae family. Here, we show that the full substitution of spermine in chicory (Cichorium intybus) requires the successive action of two enzymes, that is spermidine hydroxycinnamoyl transferase-like proteins 1 and 2 (CiSHT1 and CiSHT2), two members of the BAHD enzyme family. Deletion of these genes in chicory using CRISPR/Cas9 gene editing technology evidenced that CiSHT2 catalyzes the first N-acylation steps, whereas CiSHT1 fulfills the substitution to give rise to tetracoumaroyl spermine. Additional experiments using Nicotiana benthamiana confirmed these findings. Expression of CiSHT2 alone promoted partially substituted spermine accumulation, and coexpression of CiSHT2 and CiSHT1 promoted synthesis and accumulation of the fully substituted spermine. Structural characterization of the main product of CiSHT2 using nuclear magnetic resonance revealed that CiSHT2 preferentially catalyzed N-acylation of secondary amines to form N5,N10-dicoumaroyl spermine, whereas CiSHT1 used this substrate to synthesize tetracoumaroyl spermine. We showed that spermine availability may be a key determinant toward preferential accumulation of spermine derivatives over spermidine derivatives in chicory. Our results reveal a subfunctionalization among the spermidine hydroxycinnamoyl transferase that was accompanied by a modification of free polyamine metabolism that has resulted in the accumulation of this new phenolamide in chicory and most probably in all Asteraceae. Finally, genetically engineered yeast (Saccharomyces cerevisiae) was shown to be a promising host platform to produce these compounds.

Inactivation of the germacrene A synthase genes by CRISPR/Cas9 eliminates the biosynthesis of sesquiterpene lactones in Cichorium intybus L.

Chicory (Cichorium intybus var. sativum) is an industrial crop species cultivated for the production of a fructose polymer inulin, which is used as a low-calorie sweetener and prebiotic. Besides, inulin chicory taproots also accumulate sesquiterpene lactones (STLs). These are bitter tasting compounds, which need to be removed during inulin extraction, resulting in additional costs. In this work, we describe chicory lines where STL accumulation is almost completely eliminated. Genome editing using the CRISPR/Cas9 system was used to inactivate four genes that encode the enzyme that performs the first dedicated step in STL synthesis, germacrene A synthase (CiGAS). Chicory lines were obtained that carried null mutations in all four CiGAS genes. Lines lacking functional CiGAS alleles showed a normal phenotype upon greenhouse cultivation and show nearly complete elimination of the STL synthesis in the roots. It was shown that the reduction in STLs could be attributed to mutations in genetically linked copies of the CiGAS-short gene and not the CiGAS-long gene, which is relevant for breeding the trait into other cultivars. The inactivation of the STL biosynthesis pathway led to increase in phenolic compounds as well as accumulation of squalene in the chicory taproot, presumably due to increased availability of farnesyl pyrophosphate (FFP). These results demonstrate that STLs are not essential for chicory growth and that the inhibition of the STL biosynthesis pathway reduced the STL levels chicory which will facilitate inulin extraction.

Comparison of carbohydrate partitioning and expression patterns of some genes involved in carbohydrate biosynthesis pathways in annual and biennial species of Cichorium spp.

Variation in metabolism and partitioning of carbohydrates, particularly fructans, between annual and perennial Cichorium species remains a challenging topic. To address this problem, an annual (endive, Cichorium endive L. var. Crispum; Asteraceae) and a biennial species (chicory, Cichorium intybus L. var. Witloof; Asteraceae) were compared with in terms of variability in carbohydrate accumulation and expression patterns of fructan-active enzyme genes, as well as sucrose metabolism at various growth and developmental stages. In general, constituents such as 1-kestose, nystose, and inulin were detected only in the root of chicory and were not present in any of the endive tissues. For both species, flower tissue contained maximum levels of both fructose and glucose, while for sucrose, more fluctuations were observed. On the other hand, all the genes under study exhibited variation, not only between the two species but also among different tissues at different sampling times. In endive root compared to endive leaf, the expression of cell wall invertase genes and sucrose accumulation decreased simultaneously, indicating the limited capacity of its roots to absorb sucrose, a precursor to inulin production. In addition, low expression of fructan: fructan fructosyltransferase in endive root compared to chicory root confirmed the inability of endive to inulin synthesis. Overall, annual and biennial species were different in the production of inulin, transport, remobilization, and unloading of sucrose.

Asparagine accumulation in chicory storage roots is controlled by translocation and feedback regulation of asparagine biosynthesis in leaves.

The presence of acrylamide (AA), a potentially carcinogenic and neurotoxic compound, in food has become a major concern for public health. AA in plant-derived food mainly arises from the reaction of the amino acid asparagine (Asn) and reducing sugars during processing of foodstuffs at high temperature. Using a selection of genotypes from the chicory (Cichorium intybus L.) germplasm, we performed Asn measurements in storage roots and leaves to identify genotypes contrasting for Asn accumulation. We combined molecular analysis and grafting experiments to show that leaf to root translocation controls Asn biosynthesis and accumulation in chicory storage roots. We could demonstrate that Asn accumulation in storage roots depends on Asn biosynthesis and transport from the leaf, and that a negative feedback loop by Asn on CiASN1 expression impacts Asn biosynthesis in leaves. Our results provide a new model for Asn biosynthesis in root crop species and highlight the importance of characterizing and manipulating Asn transport to reduce AA content in processed plant-based foodstuffs.

In-vitro and in-vivo antioxidant assays of chicory plants (Cichorium intybus L.) as influenced by organic and conventional fertilisers.

BACKGROUND: Chicory (Cichorium intybus L.) is a traditional European crop that is highly appreciated for its contents of bioactive compounds, especially phenolics, which have high antioxidant activities. Among other factors, agricultural practice might affect the contents of these bioactive compounds, which are also important from a nutritional point of view, and affect the shelf-life. RESULTS: The antioxidant potential (AOP) of chicory plants treated with different fertilisers was investigated in vitro using DPPH radical scavenging and in vivo using the yeast Saccharomyces cerevisiae. Additionally, total phenolics content (TPC) was evaluated using Folin-Ciocalteu reagent, and total flavonoids content (TFC) using the aluminium chloride method. Four different chicory cultivars were included: 'Treviso', 'Verona' and 'Anivip' as red cultivars; and 'Castelfranco' as a red-spotted cultivar. These were grown in pots under controlled glasshouse conditions using organic and/or mineral fertilisers. The combination of organic and mineral fertilisers during red chicory growth resulted in significantly higher in-vitro and in-vivo AOPs compared to the control. For the red-spotted cultivar 'Castelfranco', this combined organic and mineral fertilisation decreased AOPs in vitro and increased AOPs in vivo. Among the cultivars examined, 'Castelfranco' treated with combined organic plus mineral fertilisers showed the highest AOP in vivo, accompanied by the lowest TPC and TFC. CONCLUSIONS: These data show that application of different fertilisers has different impacts on red and red-spotted chicory cultivars in terms of TFC and TPC, which for red-spotted chicory resulted in different AOPs in vitro and in vivo. The in-vitro AOP is well reflected in the in-vivo AOP for the red chicory cultivars, but less so for the red-spotted cultivar 'Castelfranco'. Based on the in-vivo AOPs for these chicory cultivars analysed, the combined organic plus mineral fertiliser treatment is recommended.

Insights into sucrose pathway of chicory stems by integrative transcriptomic and metabolic analyses.

The worldwide-cultivated chicory (Cichorium intybus L.) produces food and beneficial compounds, and young pre-flowering inflorescence stems are newly marketed vegetables. These sink-organs undergo growth by metabolizing sugars of leaf origin; the carbohydrate content and sweetness are crucial aspects for consumers' nutrition and acceptance. NMR profiling of 31 hydrosoluble phytochemicals showed that stem contents varied as influenced by genotype, environment and interaction, and that higher sucrose levels were associated with the sweeter of two landraces. Integrative analyses of metabolic and transcriptomic profile variations allowed the dissection of sucrose pathway. Overall, 427 and 23 unigenes respectively fell into the categories of sucrose metabolism and sugar carriers. Among 10 differentially expressed genes, the 11474/sucrose synthase, 53458/fructokinase, 9306 and 17035/hexokinases, and 20171/SWEET-type genes significantly associated to sugar content variation, and deduced proteins were characterised in silico. Correlation analyses encompassing sugar level variation, expressions of the former genes and of computationally assigned transcription factors (10938/NAC, 14712/bHLH, 40133/TALE and 17846/MIKC) revealed a gene network. The latter was minimally affected by the environment and accomplished with markers, representing a resource for biological studies and breeding.

Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory.

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated with protein CAS9) is a genome-editing tool that has been extensively used in the last five years because of its novelty, affordability, and feasibility. This technology has been developed in many plant species for gene function analysis and crop improvement but has never been used in chicory (Cichorium intybus L.). In this study, we successfully applied CRISPR/Cas9-mediated targeted mutagenesis to chicory using Agrobacterium rhizogenes-mediated transformation and protoplast transfection methods. A U6 promoter (CiU6-1p) among eight predicted U6 promoters in chicory was selected to drive sgRNA expression. A binary vector designed to induce targeted mutations in the fifth exon of the chicory phytoene desaturase gene (CiPDS) was then constructed and used to transform chicory. The mutation frequency was 4.5% with the protoplast transient expression system and 31.25% with A. rhizogenes-mediated stable transformation. Biallelic mutations were detected in all the mutant plants. The use of A. rhizogenes-mediated transformation seems preferable as the regeneration of plants is faster and the mutation frequency was shown to be higher. With both transformation methods, foreign DNA was integrated in the plant genome. Hence, selection of vector (transgene)-free segregants is required. Our results showed that genome editing with CRISPR/Cas9 system can be efficiently used with chicory, which should facilitate and accelerate genetic improvement and functional biology.

A BAHD neofunctionalization promotes tetrahydroxycinnamoyl spermine accumulation in the pollen coat of the Asteraceae family.

In eudicotyledons, accumulation of trihydroxycinnamoyl spermidine that is restricted to the pollen wall constitutes an evolutionary conserved trait. However, the role of this compound, which is synthetized by the BAHD enzyme spermidine hydroxycinnamoyl transferase (SHT), is still a matter of debate. Here, we show that this particular phenolamide is replaced by tetrahydroxycinnamoyl spermine in the pollen coat of the Asteraceae. Phylogenetic analyses combined with quantitative RT-PCR experiments allowed the identification of two homologous genes from Cichorium intybus (chicory) putatively involved in its metabolism. In vitro biochemical characterization of the two enzymes, named CiSHT1 and CiSHT2, confirmed the capability of recombinant proteins to synthesize spermine as well as spermidine derivatives. The wild-type metabolic phenotype was partially restored in an Arabidopsis sht mutant expressing CiSHT2. Strikingly, the transgenic plants also accumulated spermine derivatives that were absent in the wild-type. Overexpression of CiSHT2 in chicory hairy roots led to the accumulation of spermine derivatives, confirming its in vivo function. Complementary sequence analyses revealed the presence of an amino acid motif typical of the SHTs among the BAHD enzyme family. Our results highlight a recent neofunctionalization among the SHTs that has promoted the emergence of new phenolamides in the Asteraceae, which could potentially have contributed to the evolutionary success of this family.

Chicory Roots for Prebiotics and Appetite Regulation: A Pilot Study in Mice.

The objectives of this work are to address the prebiotic effects of chicory ( Cichorium intybus) together with its possible role in appetite control. We compared nine chicory genotypes in order to determine if variations in the content of metabolites in the roasted roots would lead to modifications in release of satiety hormones and in composition of gut microbiota. To this aim, a 5-week dietary-intervention study was achieved using mice fed with distinct chicory-based preparations. A 16S rRNA gene-based metagenetic analysis of fecal microbiota was performed. In vitro gastrointestinal digestions were performed in order to study the effect of chicory intestinal digests on gut hormone regulation in enteroendocrine cells. Firmicutes/Bacteroidetes ratio and gut bacterial groups, such as Alloprevotella, Blautia, Alistipes, and Oscillibacter, were found to be modulated by chicory. On the other hand, CCK and GLP-1 satiety hormones were demonstrated to be significantly increased by chicory in vitro.

Chicory R2R3-MYB transcription factors CiMYB5 and CiMYB3 regulate fructan 1-exohydrolase expression in response to abiotic stress and hormonal cues.

In the biennial Cichorium intybus, inulin-type fructans accumulate in the taproot during the first year. Upon cold or drought exposure, fructans are degraded by fructan exohydrolases, affecting inulin yield and degree of polymerization. While stress-induced expression of 1-FEH genes has been thoroughly explored, the transcriptional network mediating these responses has remained unknown. In this study, several R2R3-MYB transcriptional regulators were analysed for their possible involvement in 1-FEH regulation via transient transactivation of 1-FEH target promoters and for in vivo co-expression with target genes under different stress and hormone treatments. CiMYB3 and CiMYB5 selectively enhanced promoter activities of 1-FEH1, 1-FEH2a, and 1-FEH2b genes, without affecting promoter activities of fructosyltransferase genes. Both factors recognized the MYB-core motifs (C/TNGTTA/G) that are abundantly present in 1-FEH promoters. In chicory hairy root cultures, CiMYB5 displayed co-expression with its target genes in response to different abiotic stress and phytohormone treatments, whereas correlations with CiMYB3 expression were less consistent. Oligofructan levels indicated that the metabolic response, while depending on the balance of the relative expression levels of fructan exohydrolases and fructosyltransferases, could be also affected by differential subcellular localization of different FEH isoforms. The results indicate that in chicory hairy root cultures CiMYB5 and CiMYB3 act as positive regulators of the fructan degradation pathway.

CiMYB17, a stress-induced chicory R2R3-MYB transcription factor, activates promoters of genes involved in fructan synthesis and degradation.

In Cichorium intybus, inulin metabolism is mediated by fructan-active enzymes (FAZYs): sucrose:sucrose 1-fructosyltransferase (1-SST), fructan:fructan 1-fructosyltransferase (1-FFT), and fructan 1-exohydrolases 1, 2a and 2b (1-FEH1, -2a and -2b), respectively. While these enzymes have been rigorously characterized, the transcriptional network orchestrating their development- and stress-related expression has remained largely unknown. Here, the possible role of R2R3-MYB transcription factors in FAZY regulation was explored via bioinformatic identification of R2R3-MYBs (using an RNA sequencing (RNAseq) database), studies of co-expression of these factors with target genes, in vivo transient transactivation assays of FAZY target promoters (dual luciferase assay), and a yeast one-hybrid assay investigating the specificity of the binding of these factors to cis-elements. The chicory MYB transcription factor CiMYB17 specifically activated promoters of 1-SST and 1-FFT by binding to the consensus DNA-motif DTTHGGT. Unexpectedly, CiMYB17 also activated promoters of fructan exohydrolase genes. The stimulatory effect on promoter activities of sucrose transporter and cell wall invertase genes points to a general role in regulating the source-sink relationship. Co-induction of CiMYB17 with 1-SST and 1-FFT (and, less consistently, with 1-FEH1/2) in nitrogen-starved or abscisic acid (ABA)-treated chicory seedlings and in salt-stressed chicory hairy roots supports a role in stress-induced fructan metabolism, including de novo fructan synthesis and trimming of pre-existing fructans, whereas the reduced expression of CiMYB17 in developing taproots excludes a role in fructan accumulation under normal growth conditions.

A method for genotyping elite breeding stocks of leaf chicory (Cichorium intybus L.) by assaying mapped microsatellite marker loci.

BACKGROUND: Leaf chicory (Cichorium intybus subsp. intybus var. foliosum L.) is a diploid plant species (2n = 18) of the Asteraceae family. The term "chicory" specifies at least two types of cultivated plants: a leafy vegetable, which is highly differentiated with respect to several cultural types, and a root crop, whose current industrial utilization primarily addresses the extraction of inulin or the production of a coffee substitute. The populations grown are generally represented by local varieties (i.e., landraces) with high variation and adaptation to the natural and anthropological environment where they originated, and have been yearly selected and multiplied by farmers. Currently, molecular genetics and biotechnology are widely utilized in marker-assisted breeding programs in this species. In particular, molecular markers are becoming essential tools for developing parental lines with traits of interest and for assessing the specific combining ability of these lines to breed F1 hybrids. RESULTS: The present research deals with the implementation of an efficient method for genotyping elite breeding stocks developed from old landraces of leaf chicory, Radicchio of Chioggia, which are locally dominant in the Veneto region, using 27 microsatellite (SSR) marker loci scattered throughout the linkage groups. Information on the genetic diversity across molecular markers and plant accessions was successfully assessed along with descriptive statistics over all marker loci and inbred lines. Our overall data support an efficient method for assessing a multi-locus genotype of plant individuals and lineages that is useful for the selection of new varieties and the certification of local products derived from Radicchio of Chioggia. CONCLUSIONS: This method proved to be useful for assessing the observed degree of homozygosity of the inbred lines as a measure of their genetic stability; plus it allowed an estimate of the specific combining ability (SCA) between maternal and paternal inbred lines on the basis of their genetic diversity and the predicted degree of heterozygosity of their F1 hybrids. This information could be exploited for planning crosses and predicting plant vigor traits (i.e., heterosis) of experimental F1 hybrids on the basis of the genetic distance and allelic divergence between parental inbred lines. Knowing the parental genotypes would allow us not only to protect newly registered varieties but also to assess the genetic purity and identity of the seed stocks of commercial F1 hybrids, and to certificate the origin of their food derivatives.

HIGH FREQUENCY GENETIC TRANSFORMATION OF CICHORIUM INTYBUS L. USING nptII GENE AS A SELECTIVE MARKER.

Cichorium intybus L. is an important vegetable crop used as salad (leaf form) and for the production of coffee substitutes (root form). At the same time these plants can also be used in biotechnologies for synthesis of pharmaceutical proteins. Here we report the possibility of high frequency Agrobacterium rhizogenes- or A. tumefaciens-mediated transformation of C. intybus L. for construction of transgenic "hairy" roots and plants. The used plasmids contained target human interferonifn-α2b gene, Mycobacterium tuberculosis ESAT6:Ag85B antigene esxA::fbpB(ΔTMD) fused gene and human telomerase reverse transcriptase h Tert gene. Using of nptII gene as a selective one was preferable to the bar gene for chicory. In this case the frequency of transgenic plants or "hairy" roots formation was significantly higher. Cultivation of explants on the medium with Basta in concentration 1-2 mg/l have led to plants death or to significant reduction of number of shoots formed. Frequency of "hairy" roots formation varied from 5.9 to 42.3% after A. rhizogenes-mediated transformation. Frequency of regeneration of transgenic plants varied from 10 to 86% after A. tumefaciens-mediated transformation. Both A. rhizogenes- and A. tumefaciens-mediated transformation frequency depended on the type of explants, roots or cotyledons, and vector used. Usage of A. tumefaciens carrying pCB064 plasmid (target esxA:fbpB(ΔTMD) fused gene and nptII selective gene) resulted in the most effective regeneration of transgenic plants with regeneration frequency up to 86%. In the case of chicory A. rhizogenes-mediated transformation the highest regeneration frequency up to 42.3% was demonstrated using p CB161 vector with ifn-α2b target gene and nptII selective gene.

Effects of nuclear genomes on anther development in cytoplasmic male sterile chicories (Cichorium intybus L.): morphological analysis.

The Cichorium intybus flower development in fertile, cytoplasmic male sterility (CMS 524) and various phenotypes carrying the 524 male sterile cytoplasm was investigated macroscopically and by light microscopy. The development was similar in fertile and in male sterile florets up to meiosis, and then it was affected in anther wall structure and pollen grain development in male sterile floret. In the male sterile plants, the tapetum intrusion after meiosis was less remarkable, the microspores started to abort at vacuolate stage, the connective tissue collapsed, and endothecium failed to expand normally and did not undergo cell wall lignification, which prevented anther opening since the septum and stomium were not disrupted. Crosses undertaken in order to introduce the CMS 524 into two different nuclear backgrounds gave rise to morphologically diversified progenies due to different nuclear-mitochondrial interactions. Macroscopic and cytological investigations showed that pollen-donor plants belonging to Jupiter population had potential capacity to restore fertility while the CC line could be considered as a sterility maintainer.

Genetic diversity and relationship of chicory (Cichorium intybus L.) using sequence-related amplified polymorphism markers.

Chicory is a crop with economically important roles and is cultivated worldwide. The genetic diversity and relationship of 80 accessions of chicories and endives were evaluated by sequence-related amplified polymorphism (SRAP) markers to provide a theoretical basis for future breeding programs in China. The polymorphic rate was 96.83%, and the average polymorphic information content was 0.323, suggesting the rich genetic diversity of chicory. The genetic diversity degree of chicory was higher (GS = 0.677) than that of endive (GS = 0.701). The accessions with the highest genetic diversity (effective number of alleles, NE = 1.609; Nei's genetic diversity, H = 0.372; Shannon information index, I = 0.556) were from Italy. The richest genetic diversity was revealed in a chicory line (NE = 1.478, H = 0.289, I = 0.443) among the 3 types (line, wild, and cultivar). The chicory genetic structure of 8 geographical groups showed that the genetic differentiation coefficient (GST) was 14.20% and the number of immigrants per generation (Nm) was 3.020. A GST of 6.80% and an Nm of 6.853 were obtained from different types. This observation suggests that these chicory lines, especially those from the Mediterranean region, have potential for providing rich genetic resources for further breeding programs, that the chicory genetic structure among different countries obviously differs with a certain amount of gene flow, and that SRAP markers could be applied to analyze genetic relationships and classifications of Cichorium intybus and C. endivia.

In vitro regeneration ability of diploid and autotetraploid plants of Cichorium intybus L.

Polyploidy has played a significant role in the evolutionary history of plants and is a valuable tool for obtaining useful characteristics. Because of the novelty of polyploids, comparison of their in vitro culture response with diploids would be notable. In this study, leaf explants from diploid, autotetraploid and mixoploid plants of Cichorium intybus L. were cultured in vitro on the similar media and under same conditions. The ploidy level of the obtained calluses and regenerants were determined by flow cytometry analysis. The callogenic response of leaf explants cultured on the callus induction medium did not depend on the ploidy level of their parental plants. According to the flow cytometry analysis, the increased ploidy levels (4x) and (8x) were observed in the callus cultures with diploid and tetraploid origin, respectively. A considerable difference was observed between the ploidy level of mixoploid plants and their calluses, indicating the dominance of diploid cells in the callus tissue. The results showed that polyploidy led to the loss of organogenic potential as the tetraploid origin calluses failed to regenerate, while the diploid origin calluses successfully regenerated to whole plants.